Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.

Jastrzebska, Zaneta and Kaminska, Joanna and Chelstowska, Anna and Domanska, Anna and Rzepnikowska, Weronika and Sitkiewicz, Ewa and Cholbinski, Piotr and Gourlay, Campbell W. and Plochocka, Danuta and Zoladek, Teresa (2015) Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization. European Journal of Cell Biology, 94 (12). pp. 576-588. ISSN 0171-9335. (doi:https://doi.org/10.1016/j.ejcb.2015.10.005) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://doi.org/10.1016/j.ejcb.2015.10.005

Abstract

Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28 °C and did not grow at 37 °C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.

Item Type: Article
Uncontrolled keywords: Yeast; Rsp5 Ubiquitin ligase; Phosphorylation; Protein kinase A
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Sue Davies
Date Deposited: 16 Feb 2016 15:19 UTC
Last Modified: 17 Feb 2016 09:36 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/54221 (The current URI for this page, for reference purposes)
  • Depositors only (login required):